Trends in Food Science & Technology xxx (2017) xxx-xxx

Contents lists available at ScienceDirect

Trends in Food Science & Technology

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journal homepage: www.elsevier.com

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Review

“New trends for a classical enzyme: Papain, a biotechnological success story in the
food industry”
Jesús Fernández-Lucasa, b, ∗, Daniel Castañedab, Daniel Hormigoa, ∗∗

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a
Applied Biotechnology Group, Department of Pharmacy and Biotechnology, School of Biomedical Sciences, Universidad Europea de Madrid, Urbanización El Bosque, Calle
Tajo s/n, 28670 Villaviciosa de Odón, Madrid, Spain
b
Grupo de Investigación en Desarrollo Agroindustrial Sostenible, Department of Agroindustrial Engineering, School of Environmental Sciences, Universidad de la Costa, Cra. 55
#58-66, Barranquilla, Colombia

ARTICLE INFO ABSTRACT

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Article history: Background
Received 4 April 2017 In recent years, proteases have arisen as standard biocatalysts in many industrial processes in different
Received in revised form 19 August 2017
Accepted 23 August 2017
Available online xxx

Keywords:
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fields, such as pharmaceutical, medicine, detergent manufacturing and food science. Among them, papain is
undoubtedly one of the most frequently studied and widely used proteases in the food industry around the
world. However, the latest advances in recombinant papain expression systems, genetically engineered biocat-
alysts, new purification and isolation strategies, and enzymatic immobilization will enhance the development
of new applications of papain, as well as improve and optimize classical applications.
Cysteine proteases Scope and approach
Papain This review addresses not only the latest advances in classic applications, such as meat tenderization and
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Enzyme biotechnology protein hydrolysates, but also the most innovative applications in different industries such as food, animal
Biocatalysis feed, bioactive peptides production, water treatment, baking and brewing, among many others.
Immobilization
In addition, papain is a perfect example of a successful industrial enzyme that covers all the steps of the
Industrial processes
biocatalytic cycle that are necessary for the industrial implementation of any biocatalyst. This cycle includes
the production and extraction of the enzyme concerned (from natural or recombinant sources), functional and
structural characterization, genetic improvement, immobilization and, finally, industrial application. This re-
view describes the complete biocatalytic cycle of papain.
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Key findings and conclusions

Papain is clearly a case of industrial and commercial success over the last 40 years. The key to this success
has been continual biotechnological and process engineering innovation, which has opened up a new range
of possibilities for this exciting biocatalyst. However, further efforts are needed in protein engineering and
characterization of new mutants to reach the full potential of this enzyme.
© 2017.
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1. Introduction teases are important enzymes in living organisms, being involved in

Within the global industrial enzymes market, the food industry
enzymes sector is the major segment and is expected to grow from
nearly $1.5 billion in 2016 to $1.9 billion in 2021 (BCC Research
Biotechnology Report, 2011. Enzymes in industrial applications:
Global markets).
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many different biological processes.
Protease enzymes are used in a large variety of applications,
mainly in the detergent and pharmaceutical industries, followed by the
food industry. Since proteases represent more than 60% of the enzyme
market share, with an expected CAGR (compound annual growth rate)
of 5.3% from 2014 to 2019, they are the most important type of com-

Proteases (also called peptidases or proteinases) are enzymes that
hydrolyse the peptidic linkages in protein into shorter fragments (pep-
tides) and eventually into their components, amino acids. Pro
∗
Corresponding author. Applied Biotechnology Group, Department of Pharmacy and
Biotechnology, School of Biomedical Sciences, Universidad Europea de Madrid,
Urbanización El Bosque, Calle Tajo s/n, 28670 Villaviciosa de Odón, Madrid, Spain.
∗∗
Corresponding author.
Email addresses: jesus.fernandez2@universidadeuropea.es (J. Fernández-Lucas);
daniel.hormigo@universidadeuropea.es (D. Hormigo)
mercialized enzymes in the world. Leading producers worldwide in-
clude Novo Industries, DSM, DuPont Industries, BASF, Genencor In-
ternational, and Roche (Feijoo-Siota & Villa, 2011; Kumar, Singh,
Sangwan, & Gill, 2014).
According to the nature of the active site, proteases can be divided
into seven mechanistic classes: serine proteases, S (EC 3.4.21); cys-
teine proteases, C (EC 3.4.22); aspartic and glutamic proteases, D or
E (EC 3.4.23); metalloproteases, M (EC 3.4.24); threonine proteases,
T (EC 3.4.25); and asparagine peptide lyases, N (EC 4.3) (). Accord-
ing to the MEROPS database, cysteine proteases are divided into ten
clans, and most plant cysteine proteases belong to the C1 family, also

https://doi.org/10.1016/j.tifs.2017.08.017
0924-2244/© 2017.
2 Trends in Food Science & Technology xxx (2017) xxx-xxx
known as the papain family (papain-like proteases) Rawlings et al.,
2010). Papain-like proteases are found in most known organisms, such
as viruses, protozoa, plants, invertebrates, and vertebrates. Most of
the C1 family members are endopeptidases, but some of them dis-
play a wide variety of activities, including aminopeptidases, dipeptidyl
peptidases and enzymes with both exo- and endo-peptidase activities
(Feijoo-Siota & Villa, 2011; Novinec & Lenarčič, 2013; Rawlings et
al., 2010).
Papain (EC 3.4.22.2), also called Papaya proteinase I (PPI), is a
23.4 kDa, 212 residue cysteine endopeptidase belonging to subfamily
C1A of papain-like proteases (Rawlings et al., 2010). The commer-
cial importance of papain is mainly due to its strong proteolytic activ-
ity against a broad range of protein substrates, and because it is active
across a broad range of operational conditions. These interesting char-
acteristics have allowed papain to lead the proteases market, outselling
other plant derived proteases, such as bromelain (Ananas comosus)
and ficin (Ficus carica), as well as fungal source proteases (Market
Research Future, 2017. Global Meat Tenderizing Agents Market Re-
search Report- Forecast to 2023). Major producers of papain include
India, Sri Lanka, Democratic Republic of Congo, Zaire, Tanzania,
Uganda, Mexico, Brazil and Argentina. As regards the global papain
market, the main importers are concentrated in Europe and USA, with
a market size of about 150–200 and 300–400 tons per year, respec-
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tively. Furthermore, the Japanese market is relatively small (less than
50 tons per year). Some of the main limiting factors of the global and
sustainable supply of papain are the high climatic dependence of pa-
paya crops, as well as the economical and political issues of some pro-
ducing countries (IDEA, 2000. Commercialisation Bulletin 13 Papain
Report). This fact makes the search for alternative sources of papain
a priority for producing companies. Fortunately, the latest advances in
recombinant papain expression may provide a solution to this prob-
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lem.
This review presents papain as an industrial success paradigm in
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Biocatalysis, where all the steps necessary for the industrial imple-
mentation of any enzyme have been effectively addressed (Fig. 1).
The review provides a detailed description of each step, including the
different strategies of isolation and purification from natural and re
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Fig. 1. Complete biocatalytic cycle of papain.
combinant sources, operational conditions, genetic modifications to
improve its functional characteristics, enzyme immobilization, and in-
dustrial applications. It also describes the most recent trends in meat
tenderization, dairy industry, production of protein hydrolysates and
bioactive peptides, food allergens removal, brewing and baking indus-
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try, animal feed and water treatment, among other fields. Finally, we
look at some industrial uses of papain not strictly focused on the food
industry, but which demonstrate the enormous versatility of this ex-
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citing enzyme, such as certain biomedical approaches, antimicrobial
food packaging, caries removal agent, or tooth-whitening additive in
toothpastes.
2. Production and purification strategies
2.1. Plant derived papain
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Papain can be obtained from the latex of the papaya plant (Car-
ica papaya), which is a natural source of other endopeptidases, such
as chymopapain (EC 3.4.22.6), caricain (EC 3.4.22.30) and glycyl
endopeptidase (EC 3.4.22.25). In fact, papain is a minor constituent
(5–8%) among the papaya endopeptidases (Feijoo-Siota & Villa,
2011).
Purification of papain from papaya latex has traditionally been car-
ried out using precipitation methods, reaching high yields of up to 53 g
of crude enzyme per kg of latex. Although these methods have rou-
tinely been used in industry, they can only achieve up to 39% purity
of papain (Nitsawang, Hatti-Kaul, & Kanasawud, 2006). An alterna-
tive and more efficient purification strategy involves the use of various
chromatographic techniques (ion exchange or affinity). In these types
of techniques the initial pre-processing of the latex is essential before
samples can be applied on a chromatography column. This processing
consists in a sun- or spray-drying process after latex extraction from
plant (Feijoo-Siota & Villa, 2011).
Current techniques usually include aqueous two-phase systems
(ATPS). These systems may be composed of two polymers in aque-
ous solution, or one polymer and salt in aqueous solution. ATPS tech-
niques have shown great potential for downstream processing of pa-
pain and other proteases, since they allow clarification, concentra
 Trends in Food Science & Technology xxx (2017) xxx-xxx 3

tion and purification of the target product in a one-pot process
(Nitsawang et al., 2006). In this respect, it is worth mentioning that
polymer–salt–water systems have aroused greater interest in the indus-
try, since they are cheaper and show less viscosity than polymer–poly-
mer–water systems. In this regard, polyethylene glycol–phosphate
Eukariotic expression systems have also been studied for produc-
tion of recombinant papain. Vernet et al. (1990) obtained 0.3 mg/L
of soluble papain by using a baculovirus/insect expression system.
Higher concentrations of 1.7 mg/L have been reported by Ramjee,
Petithory, McElver, Weber, and Kirsch (1996) using the yeast Sac-

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based systems are the most employed (Rocha & Nerli, 2013). Recent charomyces cerevisiae as expression system. More recently, Werner,
studies have demonstrated that the use of alginate as macro-ligand in Hirth, Rupp, and Zibek (2015) described the expression of a
PEG based systems improves papain purification. In a recent study, codon-optimized His-tagged papain in Pichia pastoris. After purifi-

Rocha et al. (2016) described a PEG-citrate buffer system able to pu-
rify papain from latex with a 20% of recovery. They observed that
by adding alginate (0.1% w/w) and CaCl2 they could recover up to
72% of papain and recycle PEG for purification. This strategy is very
promising, since it is low cost, easy to scale up, accurate and environ-
mentally friendly (Rocha et al., 2016).
In 2014, He et al. reported an efficient method of large scale pa-
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cation 463 mg/L enzyme can be obtained, matching the highest pro-
duction reported to date using E. coli as host (400 mg/L) (Choudhury
et al., 2009). Moreover, this codon-optimized papain can be purified
in a single step from the culture medium, and it showed a 1.4 times
higher enzymatic activity towards the chromogenic peptide Z-pheny-
lalanine-arginine-paranitroanilide compared with a commercial pa-
pain. In this study, the authors demonstrate that the expression of pa-

pain purification from unclarified papaya juice feedstock in batch
adsorption system. In this study, the authors employed a reversed
phase expanded bed adsorption chromatography (RP-EBAC) using a
FastlineTM-10-EBAC column packed with AmberliteTM-XAD-7HP.
This technique allowed the authors to purify papain in a single oper-
ation with a purification factor of 7.04 and a purity of 75% (He, bin
Tuan Chik, & Chong, 2014).
After the purification process, crude papain usually has to be
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pain is better in P. pastoris than in E. coli system (Werner et al., 2015).
It is worth noting that the traditional process of papain production
from papaya crops (up to 53 g of crude enzyme per kg of wet latex)
takes about 11–13 months from the transplant of Carica papaya un-
til the fruit is ready to produce the latex (Kamphuis, Kalk, Swarte, &
Drenth, 1984). Comparing these data with those obtained by Werner
et al. (2015) in Pichia pastoris, it can be estimated that a small culture
in a 1000-L batch fermenter could yield 463 g of papain in as little as

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treated with reducing agents in order to protect the free cysteine thiol
groups from oxidation, preserving its protease activity. When needed,
free thiol groups of papain can be regenerated by the addition of low
molecular mass thiols, such as cysteine or dithiothreitol.
2–3 days. Taking into account this enormous difference in yields as
well as the advantages that recombinant production offers regarding
purification processes, irrespective of political, economic and climatic
factors, in our opinion the production of recombinant papain will com-

2.2. Recombinant papain

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3. Operational conditions

The high world demand of papain for industrial uses makes neces-
sary the search for alternative sources from traditional extraction from
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the latex of C. papaya. Despite the high recovery of papain from Car-
ica papaya plant material (up to 53 g of crude enzyme per kg of wet la-
tex), this natural source presents several drawbacks. First, papain only
represents about 5–8% of total cysteine proteases in the latex, which
entails an expensive and ponderous isolation strategy (Nitsawang et
al., 2006). Furthermore, isolated papain is highly susceptible to oxi-
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3.1. Functional characterization
Regarding its enzymatic activity, papain has a broad-spectrum en-
dopeptidase activity over a pH range of 5–8 and optimal temperature
of 65 °C (Polaina & MacCabe, 2007).
The specificity of papain has been extensively examined using
different substrates, such as milk, haemoglobin, collagen discs and
labelled collagen products, synthetic peptides, gelatine (You,

dation, so it must immediately be conserved at low temperatures and
direct air contact must be avoided. Another major disadvantage is the
dependence of papaya crops on external factors, such as climate, soil,
pests, etc., which avoids a continuous supply of papain.
The expression of recombinant papain in different microorgan-
ism-based systems makes it possible to overcome these drawbacks
while significantly increasing the papain production capacity. Papain
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Regenstein, & Liu, 2010), casein (Morais, Silva, Oliveira, & Silvestre,
2004), chitosane (Pan, Zeng, Foua, Alain, & Li, 2016), rice bran
protein concentrates (Ahmadifard, Murueta, Abedian-Kenari,
Motamedzadegan, & Jamali, 2016), soybean proteins (Shutov et al.,
2013), potato protein isolate (Waglay & Karboune, 2016), im-
munoglobulins including sheep IgG, rabbit IgG, chicken IgY and
fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP),

is synthesized as a pre-pro-peptide with 345 amino acid residues. This and whey proteins (α-lactalbumin and β-lactoglobulin) (Chalabi,
precursor contains a signal peptide (residues 1 to 18), a spacer peptide Khademi, Yarani, & Mostafaie, 2014), among many others (Table 1).
(19–33) and the mature enzyme (134–345). The most abundant amino
acids in papain precursor protein are glycine (35), tyrosine (28) and Table 1
valine (24), while histidine (3) and methionine (5) are present in the Optimum pH and temperature for papain with different substrates.
lowest amounts.
Substrate pH T (°C) References
Papain precursor can be cloned and expressed in Escherichia coli,
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but recombinant enzyme forms inactive inclusion bodies. However, Meat 7–8 60–65 (Bekhit et al., 2014)
they can be renatured to yields about 3 mg of active papain/L (Taylor Gelatin 7.5 56.8 (You et al., 2010)
et al., 1992). In this regard, Choudhury, Roy, Chakrabarti, Biswas, Casein 7.5 37 (Morais et al., 2004)
Chitosan 4.55 44.42 (Pan et al., 2016)
and Dattagupta (2009) described an optimized purification and refold- Rice bran proteins concentrates 8 28 (Ahmadifard et al., 2016)
ing protocol, reaching 400 mg/L of a His-tagged fusion pro-papain Soybean (β-conglycin) 5.6 30 (Shutov et al., 2013)
from E. coli. However, several problems can arise during the refold- Potato protein isolate 8 50 (Waglay & Karboune, 2016)
ing process, such as autoproteolysis, oxidation of the active site and Sheep and rabbit IgG 7 37 (Chalabi et al., 2014)
Fish IgM 5.5–8.5 37 (Chalabi et al., 2014)
formation of incorrect disulfide bonds. LTP 7.2 37 (Chalabi et al., 2014)
4 Trends in Food Science & Technology xxx (2017) xxx-xxx
According to the model proposed by Schechter and Berger (1967),
papain interacts with at least seven residues of the substrate, four
amino acid residues in N-terminal direction from the cleaved bond
(named P1-P4), and three in C-terminal direction (named P1′-P3′).
Authors also proposed seven corresponding sites on the enzyme (sites
S1-S4 and S1′-S3′) which are responsible for the recognition and
cleavage of polypeptide (Novinec & Lenarčič, 2013; Schechter &
Berger, 1967). However, three decades later, the structural analysis of
reported papain structures support this definition to some extent, and
only a few sites were confirmed in papain-like proteases (sites S2, S1
and S1′) (Turk, Gunčar, Podobnik, & Turk, 1998).
In general, papain-like proteases have broad specificity and the
major determinant is the residue in the P2 position of the substrate. In
this sense, Papain specificity is controlled by the S2 site, a hydropho-
bic core that accommodates the P2 side chain of the substrate. Due
to this, papain shows high specificity for amino acids with hydropho-
bic or aromatic side chains, such as Val, Phe and Tyr, at this position.
However, papain does not recognize Val in P1′ (Choe et al., 2006;
Groves, Coulombe, Jenkins, & Cygler, 1998; ; Novinec & Lenarčič,
2013; Turk et al., 1998).
3.2. Structural characterization
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Despite the limited sequence homology, cysteine proteases dis-
play a conserved core structure, composed of two clearly differenti-
ated interacting domains, an α-helix and a β-barrel-like that have been
termed the L- and R-domains according to their position in the stan-
dard orientation (Fig. 2) (Novinec & Lenarčič, 2013). Papain struc-
ture displays a globular protein with three disulfide bonds and two
catalytic L- and R-domains. The active site is located at the inter-
face of L- and R-domains in the form of a V-shaped cleft and is
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formed by a cysteine (Cys), a histidine (His), an asparagine (Asn)
and a glutamine residue (Gln), which are conserved in all papain-like
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proteases (Fig. 2). In the mature protein, Cys25 and His159 form an
ion pair substrate-binding pocket stabilized by Asn175, which orients
the His159 imidazolium ring. The sulfhydryl group of Cys25 performs
a nucleophilic attack on the carbonyl carbon of a substrate's peptide
bond, leading to an unstable tetrahedral intermediate. This interme-
diate spontaneously collapses to regenerate the carbonyl group, lead-
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ing to an acyl enzyme complex, which is hydrolysed into the free en-
zyme and the N-terminal portion of the substrate (Fig. 3) (Cstorer &
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Fig. 2. A) Three-dimensional structure of papain (PDB accession code 1PPN). The arrow indicates the situation of active site. B) Catalytic residues in the active site of papain. Cys25
and His159 are forming the catalytic ion pair.
Ménard, 1994). Another important residue is Gln19, which precedes
the Cys25 and is believed to help in forming an oxyanion hole by sta-
bilizing the tetrahedral intermediate. Asn175 forms a hydrogen bond to
His159 (showing a similar role to the aspartic acid in the catalytic triad
of serine proteases), but is not necessary for catalysis (Vernet et al.,
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1995). Finally, it is interesting to note that Trp177 is involved in the
generation of the nucleophilic character of Cys25/His159 ion pair (Fig.
2).
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In order to highlight the substrate binding-mode, a three-dimen-
sional scheme of all described binding interactions is shown using the
crystal structure of papain complexed with CLIK148, a representative
cathepsin L-specific inhibitor (Fig. 4) (Tsuge et al., 1999). On one
side, the hydrogen bond formed between Asn175 and His159 stabilizes
the substrate-binding pocket and also orients the His159 imidazolium
ring. On the other side, the sulfhydryl group of catalytic Cys25 is cova-
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lently bonded to the C2 atom of CLIK148. Apart from that, CLIK148
is stabilized in the active site by a network of hydrogen bonds. A hy-
drogen bond is formed between the nitrogen atom of both Cys25 and
Gln19 and the oxygen atom O1 of CLIK148. Besides, the Gly66 pep-
tide nitrogen is hydrogen bonded to the O3 atom of CLIK148. Another
hydrogen bond is formed between an oxygen atom of Asp158 and the
nitrogen atom N5 of CLIK148. As shown in Figs. 2 and 4, a water
molecule is located in the active site, near the residue His159. In this
regard, three hydrogen bonds are formed from N7 atom of CLIK148,
carbonyl oxygen of Asp158 and N1 atom of His159 to the water mole-
cule. Finally, several hydrophobic interactions among CLIK148 with
Trp177 (responsible for the stacking of pyridine ring of the substrate)
and Val133 are shown.
4. Biocatalyst improvement
4.1. Genetic modifications
As mentioned above, several studies have addressed papain mu-
tations to increase expression levels. Other studies, however, have
told us more about the catalytic mechanism of this enzyme, such as
that carried out by Gul et al. (2008), who demonstrated that Asp158
does not affect to nucleophilic character in the Cys25-His159 dyad as
previously thought, whereas Trp177 showed a key role. Choudhury,
Biswas, Roy, and Dattagupta (2010) developed single (Lys174→Arg),
double (Lys174→Arg, Val32→Ser) and triple mutants of papain
 Trends in Food Science & Technology xxx (2017) xxx-xxx 5

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Fig. 3. Catalytic mechanism of papain.

(Lys174→Arg, Val32→Ser, Gly36→Ser), increasing the thermostabil-

ity of the biocatalyst. The triple mutant was the most thermostable, in-
creasing its half-life by 45 min at 60 °C and at 65 °C compared with
wild type papain. In addition, the triple mutant had a faster inactiva-
tion rate beyond Tmax, which is very interesting for industrial imple-
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mentation.
Recent studies have demonstrated that the specificity of papain can
be modified by replacing the residue Ile86 in its pro-peptide region.
These mutations (Ile86→Phe, Ile86→Leu and Ile86→Ala) can block the
specificity determining S2-subsite of the catalytic cleft of the protease
in its zymogen form and significantly improve the macroscopic ki-
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netic parameters (Km and kcat) of the enzyme (Dutta, Choudhury, Roy,
Dattagupta, & Biswas, 2016). Several of these studies and others are
summarized in Table 2.

5. Papain immobilization

Enzyme immobilization provides an excellent base for increasing

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enzyme stability and half-life, also simplifying downstream process-

Fig. 4. Crystal structure of CLIK148 (cyan C atoms with heteroatom coloring) bound in
the active site of papain. The hydrogen bonding interactions between ligand and protein
are draw as black dashed lines. Water molecule is draw as red sphere. (For interpreta-
tion of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
ing. Thus, many different methods exist for enzymatic immobiliza-
tion (physical adsorption, entrapment, copolymerization or covalent
attachment, among others), and several natural and synthetic supports
have been assessed for their efficiency in the process. Since papain
has been utilized in a large number of industrial applications, numer-
ous efficient strategies on a variety of carriers have been described
in the current literature, such as starch gel (Sangeetha & Abraham,
2006), nitrilon fibre functionalized with amine groups (Li, Xing, &
Ding, 2007), cotton fabric (Xue, Nie, Zhu, Li, & Zhang, 2010) or
6 Trends in Food Science & Technology xxx (2017) xxx-xxx

Table 2
Mutant variants of papain.

Mutation Host Result References

Lys174→Arg Lys174→Arg/Val32→Ser E. coli Increase thermal stability (Choudhury et al., 2010)

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Lys174→Arg/Val32→Ser,Gly36→Ser
Ile86→Phe E. coli Modification of substrate specificity (Dutta et al., 2016)
Ile86→Leu
Ile86→Ala

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Gln19→Glu Baculovirus-insect cell expression system and S. Elucidation of the role of Gln19 in the active (Ménard et al., 1991)
cerevisiae site (Ménard et al., 1995)
Gln19→His
Gln19→Asn/Ser21→Ala
Gln19→ Ala
Gln19→ Ser
Val133→Ala/Ser205→Glu Baculovirus-insect cell expression system a Modification of substrate specificity of S2 (Khouri et al., 1991)
Val133→Ala/Val157→Gln/Ser205→Glu
Gly19→Glu Baculovirus-insect cell expression system Nitrile hydratase activity into papain (Dufour, Storer, & Ménard,

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1995)

sepharose (Homaei, Sajedi, Sariri, Seyfzadeh, & Stevanato, 2010), be-
tween many others.
More recent papain immobilization strategies have been included:
generation of porous cross-linked enzyme aggregates (p-CLEAs)
(Wang et al., 2011); the use of iron oxide magnetic nanoparticles
6.1. Meat tenderization
The use of exogenous proteases to improve meat tenderness has
become an increasing focus of interest recently. It is a priority for the
meat industry to be able to cover the increasing demand for guaran-

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(g-Fe2O3/Fe3O4) activated with thiophene and triazole as enzyme link-
ers (Xin, Si, & Xing, 2010); encapsulation in biosilica matrix through
a biomimetic silicification process induced by papain (Zhou, Wang,
Jiang, & Gao, 2013); magnetic gold nanocomposites modified with
3-(mercaptopropyl) trimethoxy silane (MPTS) (Sahoo, Sahu,
Bhattacharya, Dhara, & Pramanik, 2013) (Fig. 5); and crosslinking
with a glutaraldehyde to poly (vinyl alcohol) PVA nanofibers prepared
by electrospinning (Moreno-Cortez et al., 2015).
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6. Industrial applications of papain
Functional properties of papain have developed an increasing in-
terest in a wide range of industrial uses, mainly in meat tenderization,
foods, feeds, brewing and the textile industry. Papain has also been
described as an active additive in tooth-bleaching dentifrices and skin
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teed tender meat and give added value to lower-grade meat cuts.
Many approaches have been based on improving post-mortem ten-
derness, such as mechanical tenderization, water content enhance-
ment, and different enzymatic treatments (Pietrasik & Shand, 2011).
Traditionally, meat is softened by autoproteolysis (mainly mediated
by cathepsins and capains), keeping it at 4 °C for 7–10 days.
Under optimal operating conditions (pH around 7–8 and tempera-
ture between 60 and 65 °C) papain is able to hydrolyse almost any pro-
tein present in muscle tissue, as well as tendons and ligaments, which
makes it a potent meat softener (Bekhit, Hopkins, Geesink, Bekhit, &
Franks, 2014).
There are numerous studies in which the meat softening effect of
papain has been evaluated when enzyme is injected into the animal
before being slaughtered, allowing a homogenous distribution of pa-
pain in the meat of the animal. Today, this technique presents ethi-
cal concerns and is not used, since the injection of active papain can

products. This part of the review summarises the main applications of cause suffering and stress in animals. An alternative is the injection
this enzyme (Table 3). of inactive papain. For this purpose, papain is usually treated with hy
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Fig. 5. Schematic representation of the immobilization of papain on magnetic gold nanoparticles.

 Trends in Food Science & Technology xxx (2017) xxx-xxx 7

Table 3 Post-mortem application is generally acceptable for lower-grade

Industrial applications of papain. meat cuts. Papain is supplied commercially in powder and liquid
Application Mode of action References forms (e.g. PANOL®, LIQUIPANOL® T100), as well as combined
with other proteases such as bromelain (e.g., ENZECO® DUAL PRO-
Meat Hydrolysis of connective tissue and myofibrillar (Pietrasik & TEASE). Several commercial preparations may include other ingredi-

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tenderization proteins. Shand, 2011)
(Bekhit et al.,
ents (salt, phosphates or flavour enhancers such as sodium glutamate),
2014) and they are available to be used directly by processors and house-
Production Hydrolysis of proteins to form peptides of varying (Polaina & holds.

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of protein sizes with multiple applications: nutritional MacCabe,
hydrolysates supplement, pharmaceutical ingredient, flavor 2007)
6.2. Dairy industry
enhancer, bioactive properties, etc. (Agyei &
Danquah,
2011) Due to their ability to hydrolyse the specific peptide bonds to gen-
(Liu et al., erate casein and macropeptides, proteases have been extensively used
2016) as biocatalysts in milk clotting processes in the dairy industry. One of
(Meinlschmidt
et al., 2016)
the most important uses of proteases is cheese manufacturing. Tradi-

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Dairy Production of semisoft cheese or cream cheese, (Mahajan & tionally, dried calf stomachs or dried enzyme extracts (animal rennet)
industry improve the meltability and stretchability of Chaudhari, have been employed for better controlled cheese making. Due to lim-
cheese 2014) (Hejazin ited availability of calf stomachs, it was necessary to find new pro-
& El-Qudah, teases with similar properties, in order to be used as biocatalysts in
2009)
(Abe et al., the industrial production of cheese. The potential of plant proteases in
2015) cheese making has been reported in this regard (Jacob, Jaros, & Rohm,
Baking Increase of protein solubility, and reduction of (Polaina & 2011).
industry allergenic protein content of cereals. MacCabe, Arlene, Prima Kristijarti, and Ardelia (2015) studied the effect of
2007)

D
(Kong et al.,
papain in the production of Cheddar cheese with different types of
2007) milk (cow, goat and soy), concluding that papain displayed highly pro-
(Li et al., teolytic activity on milk but with a very irregular clotting performance.
2016) These results suggest that papain is not a good biocatalyst for the pro-
Animal feed Estimation of protein degradation in ruminant
feed. Production of bioactive peptides
(Polaina &
MacCabe,
2007)
(Choi et al.,
2016)
TE duction of hard cheese. However, papain shows significant advantages
over other proteases, such as better accessibility, lower price, greater
availability in large quantities, and more resistance to extreme pH and
temperatures. In this respect, if clotting performance could be con-
(Mo et al., trolled, papain could be used as a rennet enzyme replacement for the
2016)
production of hard cheese.
EC
Brewing and Degradation of several insoluble protein (Polaina &
wine aggregates formed during and after beer MacCabe, On the contrary, Mahajan and Chaudhari (2014) report the use of
industry fermentation, wine stabilization agent. 2007) papain in the production of semisoft cheese or cream cheese. Hejazin
(Esti et al., and El-Qudah (2009) improved the meltability and stretchability of
2013) Nabulsi cheese by papain addition. Finally, an interesting article de-
Bioethanol Papain acts as a deflocculating agent avoiding (Silva et al.,
industry yeast flocculation during fermentation. 2015) scribes how Abe, Wu, Kim, Fujii, and Abe (2015) developed a new
Water Binding of heavy metals, such as mercury, due to (Metin & Alver, method for soymilk cream production using a simple three-step
RR

treatment presence of four sulfhydryl groups on papain 2016) process, including papain digestion, heat treatment and low-speed cen-
active site. trifugation.
Tooth Removal of stains, plaque, and food debris from (Münchow et
whitening the outer tooth surface. al., 2016)
(Chakravarthy 6.3. Production of protein hydrolysates
& Acharya,
2012) Protein hydrolysates are widely used in industry with many dif-
Biomedicine Treatment of burn wounds, esophageal (Anuar, Zahari, ferent applications: nutritional supplement, pharmaceutical ingredi-
CO

obstruction, caries removal, activity against Taib, &

gastrointestinal nematodes, tissue repairing of Rahman, ent, flavor enhancer, nitrogen source for growth media for microbial,
venous ulcers, antibacterial activity, etc. 2008) plant, and animal cell culture, in cosmetics and in beverages. Cur-
(Bussadori et rently, the industrial production of protein hydrolysates includes acid,
al., 2014) alkali, and enzyme hydrolysis, among which the latter-mentioned is
(Ribeiro et al.,
2015)
acquiring the most importance in the food and pharmaceutical indus-
(Morse et al., tries. The enzymatic process is milder and facilitates control of the
2016) degree of hydrolysis, which makes the process efficient and repro-
UN

ducible. The operational parameters for enzymatic hydrolysis have

drogen peroxide to oxidize the catalytic cysteine and then injected into
the animal. Once the animal is sacrificed, the anoxic conditions in-
duce the reduction of catalytic cysteine and thus the reactivation of pa-
pain (Bekhit et al., 2014). However, this ante-mortem method presents
some drawbacks, mainly due to the difficulty in predicting the level of
tenderization, since this depends on different physiological factors of
the animal. Some problems that may appear are related to differences
in texture as compared with high quality meat slices, over-tenderiza-
tion, undesired tastes or smells, or degradation of organs that may be
of commercial interest.
been extensively studied, the most important being temperature, hy-
drolysis time, pH, and degree of hydrolysis (DH) (Agyei & Danquah,
2011).
As a result of hydrolysis, the parent protein forms peptides of vary-
ing sizes depending on the enzyme employed and the operational pa-
rameters. The peptides with a positive effect on health are termed
bioactive peptides, and they can regulate several physiological func-
tions. Many bioactive qualities of protein hydrolysates with papain
8 Trends in Food Science & Technology xxx (2017) xxx-xxx
have been widely studied, such as antitumor, antioxidant, antidiabetic,
inhibition of angiotensin–I converting enzyme, modulation of immune
system and mineral binding ability (Nesse, Nagalakshmi, Marimuthu,
& Singh, 2011).
Due to their high antioxidant properties, fish protein hydolysates
(FPH) have aroused special interest (Elavarasan & Shamasundar,
2016; Gajanan, Elavarasan, & Shamasundar, 2016). An important as-
pect of industrial production of FPH is related to the concentration
methods that can be employed, such as oven, freeze or spray dry-
ing. Among them, freeze drying is the preferred operation in industry,
since it causes less damage to peptides. However, high capital and run-
ning costs have led to a search for alternate drying methods. Recently,
Elavarasan and Shamasundar (2016) compared the effect of oven- and
freeze-drying on the antioxidant properties of FPH with papain from
Cirrhinus mrigala, suggesting that oven drying may be advantageous.
Moreover, enzymatic conversion of fish frame waste to protein hy-
drolysate could be a solution for minimizing the pollution issues re-
lated to seafood processing operations, and a way to add value to pro-
cessing by-products.
Another recent application of papain for the production of an-
tioxidant hydrolysates is the Chinese walnut (Juglans regia L.) hy-
drolysates (Liu et al., 2016). The authors hydrolysed walnut proteins
with different proteases including papain. This study revealed that hy-
D
drolysates obtained with papain showed a yield and purity of 16% and
81%, respectively, higher than others. Moreover, approximately 99%
of peptides had a molecular weight lower than 1500 Da. Furthermore,
the bioassay indicated that peptides obtained with papain exhibited
one of the highest antioxidant properties.
of soy protein isolates (SPI), while sensory, technical and functional
properties can be improved. It is worth mentioning that papain proved
TE
In addition, papain can be used to reduce the level of allergenicity
to be the most suitable protease for improving functionality and sen-
sory characteristics, effectively reducing the molecular weight of SPI
EC
(Meinlschmidt, Sussmann, Schweiggert-Weisz, & Eisner, 2016).
Another interesting application of papain in the production of pro-
tein hydrolysates is reported by Damodaran (2007). In this work, the
author studied the inhibition of ice crystal growth in ice creams by em-
ploying gelatine hydrolysates, previously digested with papain. These
results are very important, because understanding the molecular inter-
RR
actions responsible for ice crystal growth inhibition by peptides from
gelatine hydrolysate would greatly facilitate the development of new
peptide cryoprotective agents with enhanced antifreeze activity.
6.4. Baking industry
Wheat is one of the most harvested cereals throughout the world.
CO
Nevertheless, wheat contains proteins able to cause allergic reactions
in 0.4–1.3% of children and 0.2–0.9% of adults.
Wheat grain contains different allergenic proteins such as gliadins
(a, b, c and x), that conserve the allergenic epitope
Gln-Gln-Gln-Pro-Pro. Papain has been used to produce hypoaller-
genic flour suitable for allergic consumers, since it is able to recog-
nize this -Gln-Pro rich motif. Li, Yu, Goktepe, and Ahmedna (2016)
UN
described a complete removal of gliadins of wheat flour by a sequen-
tial enzymatic treatment with alcalase and papain. This treatment was
found to be more effective in reducing allergenic protein content and
increasing solubility compared with other proteases (Li et al., 2016).
Another issue related to gluten in the baking industry is that it
turns insoluble and expands to form lattice-like structures when it is
hydrated. Therefore, gluten should be hydrolysed to obtain more
mouldable dough. The use of proteases such as papain improves the
quality of the dough, preventing it from contracting, and enhancing its
solubility, softness and rising during baking process (Kong, Zhou, &
Qian, 2007; Polaina & MacCabe, 2007).
F
6.5. Brewing and wine industry
OO
Papain is a very commonly used enzyme in the brewing industry,
especially in the production of light and clear beers, due to their high
chill-proofing potential (Polaina & MacCabe, 2007). The treatment
of beer with papain (among other proteases) allows the degradation
of several insoluble protein aggregates formed during and after beer
fermentation, without any side effects on its organoleptic properties.
These high protein levels are responsible for several undesired effects
PR
in beer, such as hazes, high viscosity and excess foaming.
In addition, due to the broad substrate specificity displayed by pa-
pain, this protease could find useful application in winemaking. In this
respect, several reported studies have tested the effect of papain as a
wine stabilization agent. In a recent study, Esti, Benucci, Lombardelli,
Liburdi, and Garzillo (2013) evaluated papain activity under wine-like
conditions, showing that this protease could be applied efficiently as a
biocatalyst in the wine industry.
6.6. Animal feed
Approximately 90% of food energy and nutrients for ruminants is
provided by low-cost forages. Therefore, quantification of soluble ni-
trogenous compounds in the rumen is essential to evaluate the quality
of feed. Adding proteases like papain to animal feed can increase pro-
tein assimilation and bio-availability, which translates into significant
economic savings (Polaina & MacCabe, 2007).
Along these lines, the Hong Kong Agriculture, Fisheries and Con-
servation Department has employed papain to hydrolyse proteins of
soy meal for fish feed. In this study, they supplement traditional pel-
lets with soybean hydrolysates and use them for feed three different
species of marine fishes (Rhabdosargus sarba, Epinephelus bleekeri
and Trachinotus blochii). After 340 days of treatment, they observed
more relative weight gain and better growth yield in fishes fed with
papain treated pellets (Mo, Lau, Kwok, & Wong, 2016). Moreover,
Choi, Lam, Mo, and Wong (2016) used mixtures of papain and brome-
lain to hydrolyse food wastes employed to make fish feed (final com-
position of proteases at 1–2%). They observed that the use of feeds
supplemented with these mixtures of proteases improved the growth,
lipid accumulation and immunity of grass carp (Ctenopharyngodon
idella).
6.7. Other industrial applications
Due to papain's strong proteolytic activity, it has been employed as
a versatile tool in the food industry, but it has many other application
fields, such as biomedicine, dental or textile industry, among others.
Here we want to report some industrial uses of papain not strictly fo-
cused on the food industry, but which reveal the enormous versatility
and commercial potential of this enzyme.
Applications of papain in medicine over the last five years include
the treatment of proteinaceous esophageal food impaction using pa-
pain and other proteolytic enzymes as an initial treatment for all pa-
tients with esophageal obstruction (Morse, Wang, Donahue, Garrity,
& Allan, 2016); the treatment of mild to moderate acne with a fixed
combination of 0.1% hydroxypinacolone retinoate (synthetic ester of
9-cis-retinoic acid), 1% retinol in glycospheres and 2% papain in gly
 Trends in Food Science & Technology xxx (2017) xxx-xxx
cospheres in aqueous gel (Veraldi et al., 2015); and tissue repairing of
venous ulcers employing low concentrated papain gels (Ribeiro et al.,
2015).
Nowadays, the use of papain is increasingly common in the dental
industry, where the proteolytic and antibacterial properties of papain
seem to be an efficient and safe alternative for caries removal before
restorative procedures are undertaken.
Papain has also been employed as a chemo-mechanical caries re-
moval agent (CMCR), first commercialized in 2003 as Papacarie,
which is a mixture of papain, chloramine, toluidine blue and salts
in a thickening emulsion (Bussadori et al., 2014). Other similar
chemo-mechanical caries removing agents based on papain are
Apacaries (Juntavee et al., 2014), papEdent (Subramaniam &
Gilhotra, 2011) and CarieCare (Venkataraghavan et al., 2013).
One the other hand, papain has extensively been described as an
active ingredient in dentifrices. There are numerous types of tooth-
paste containing various enzymes in order to remove stains from teeth
(Chakravarthy & Acharya, 2012). More recently, Münchow, Hamann,
Carvajal, Pinal, and Bottino (2016) also described the stain removal
effect of papain- and bromelain-based dentifrices applied to enamel.
Another interesting example is the use of papain in antimicro-
bial food packaging. Cynthya, Prabhawathi, and Mukesh (2014) im-
mobilized papain on polyurethane films to avoid microbial contam-
D
ination on cheese. Experimental results showed that this derivative
could be used as effective agent control and reduce the growth of
Staphylococcus aureus biofilm formation. In the same way, Manohar,
Prabhawathi, Sivakumar, and Doble (2015) tested the antimicrobial
effect of immobilized papain derivatives against Acinetobacter spp.
and S. aureus.
Other interesting uses of papain include water treatment for re-
moval of heavy metals (Metin & Alver, 2016), or preventing yeast
TE
flocculation in the bioethanol industry (Silva, Rosa, Carvalho, &
Oliva-Neto, 2015).
EC
7. Conclusions
Papain is undoubtedly one of the most frequently studied and
widely used proteases in the industry worldwide. Despite the fact
that papain can cause an allergic response in humans (Díez-Gómez,
RR
Quirce, Aragoneses, & Cuevas, 1998; Mansfield, Ting, Haverly, &
Yoo, 1985), papain is generally recognized as safe (GRAS) as a di-
rect human food ingredient according to Code of Federal Regula-
tions of US Food and Drug Administration (U. S. Food and Drug
Administration, 2016). Moreover, according to the FAO/WHO food
standards, papain is a food additive that may be used in the foods un-
der the conditions of good manufacturing practices (GMP) as outlined
CO
in the Preamble of the Codex GSFA (FAO/WHO Codex Alimentarius
Commission, 2016).
Although one cysteine protease inhibitor has been predicted to be
in the papaya genome (Paull et al., 2008) and such inhibitor is also
suspected to be stored in the papaya latex (Azarkan, El Moussaoui,
Van Wuytswinkel, Dehon, & Looze, 2003), specific papain inhibitors,
which might compromise enzyme activity in papaya latex extracts
UN
with relevance to the food industry, have so far not been found.
Papain has been used in the food industry for decades and today
continues to dominate the world market for industrial proteases. This
is mainly due to its better operational characteristics, such as tem-
perature stability and half-life, in comparison with its main competi-
tors. However, growing market demand is outpacing supply of pa-
pain, which has boosted prices in recent years and is holding back the
9
growth of the papain market, in turn favouring the market for other
proteases such as bromelain or fungal proteases.
The latest advances in recombinant papain expression systems, es-
pecially yeast-based systems, will help overcome these difficulties and
provide the market with a cheaper and more sustainable supply of pa-
F
pain. In addition, the new strategies of purification and isolation, ge-
netic modification and enzymatic immobilization will improve the de-
velopment of new applications of papain, especially in the food indus-
OO
try, making some processes profitable, which is currently not the case
with traditional production methods.
Papain remains a very important product in the meat, brewing and
dairy industry, where the challenge is to improve the operational prop-
erties of papain and to reduce its supply. Regarding the latest trends,
most of the efforts are focused on the production of bioactive peptides
and functional foods that may have antioxidant, antitumor, or hypoal-
PR
lergenic properties.
In this respect, further efforts are needed in the functional charac-
terization of recombinant of papain, as well as in the generation of new
mutant variants of papain with improved properties. In this sense, di-
rected evolution, molecular docking, molecular dynamics and genetic
engineering could be very important tools in order to improve activity
and stability of the enzyme.
Compliance with ethical standards
Funding
This work was supported by grant SAN151610 from the Santander
Foundation.
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants
or animals conducted by any of the authors.
Acknowledgements
We thank Peter Bonney for his continued support and enthusiasm
for the project.
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